Universidade de Aveiro Ano 2018
Departamento de Biologia
DIANA ISABEL PINTO
DE ALMEIDA
STRATEGIES
TO
PROMOTE
Akkermansia
muciniphila VIABILITY AND STABILITY UNDER
STRESS CONDITIONS.
ESTRATÉGIAS
PARA
PROMOVER
A
VIABILIDADE E ESTABILIDADE DE Akkermansia
muciniphila SOB CONDIÇÕES DE STRESS.
DECLARAÇÃO
Declaro que este relatório é integralmente da minha autoria,
estando devidamente referenciadas as fontes e obras consultadas,
bem como identificadas de modo claro as citações dessas obras.
Não contém, por isso, qualquer tipo de plágio quer de textos
publicados, qualquer que seja o meio dessa publicação, incluindo
meios eletrónicos, quer de trabalhos académicos.
Universidade de Aveiro Ano 2018
Departamento de Biologia
DIANA ISABEL PINTO
DE ALMEIDA
STRATEGIES
TO
PROMOTE
Akkermansia
muciniphila VIABILITY AND STABILITY UNDER
STRESS CONDITIONS.
ESTRATÉGIAS PARA PROMOVER A
VIABILIDADE E ESTABILIDADE DE Akkermansia
muciniphila SOB CONDIÇÕES DE STRESS.
Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Microbiologia, realizada sob a orientação científica da Doutora Ana Cristina Cardoso Freitas Lopes de Freitas, Investigadora Principal do Centro de Biotecnologia e Química Fina (CBQF) – Laboratório Associado da Escola Superior de Biotecnologia, Universidade Católica Portuguesa e co-orientação da Doutora Sónia Alexandra Leite Velho Mendo Barroso, Professora Auxiliar com Agregação do Departamento de Biologia da Universidade de Aveiro e Doutora Ana Maria Pereira Gomes, Professora Associada da Escola Superior de Biotecnologia, Universidade Católica Portuguesa.
This work was supported by national funds through FCT/MEC (PIDDAC), project reference IF/00588/2015 and by Operational Program Competitiveness and Internationalization in its FEDER component and by the budget of the Foundation for Science and Technology, I.P. (FCT, IP) in its OE component, project reference POCI-01-0145-FEDER-031400 with scientific collaboration of CBQF under the FCT project UID/Multi/50016/2013.
Dedico este trabalho a toda a minha família, em especial à minha prima Andreia e tia Natália, porque a coragem nem sempre é um rugido.
“I have not failed. I’ve just found 10.000 ways that won’t work.”
Thomas Edison
“Magic happens when you don’t give up, even though you want to. The universe always falls in love with a stubborn heart.”
o júri
presidente Professora Doutora Maria Ângela Sousa Dias Alves Cunha Professora Auxiliar da Universidade de Aveiro
vogal – arguente principal
vogal – orientadora
Professor Doutor José Carlos Márcia Andrade
Professor Auxiliar do Instituto Universitário de Ciências da Saúde
Professora Doutora Ana Cristina Cardoso Freitas Lopes de Freitas Investigadora principal do Centro de Biotecnologia e Química Fina (CBQF) – Laboratório Associado da Escola Superior de Biotecnologia, Universidade Católica Portuguesa
agradecimentos À minha orientadora, Doutora Ana Freitas, pelo voto de confiança, disponibilidade, orientação, mas acima de tudo, pela amizade, paciência, incansável apoio e palavras de encorajamento que me acompanharam ao longo deste “longo” percurso.
Às minhas co-orientadoras, Doutora Sónia Mendo, pela oportunidade concedida, bem como pelo acompanhamento desta fase da minha jornada científica e Doutora Ana Gomes, pelas sessões de brainstorming, pelo input critico e científico e, pela preocupação e carinho que sempre teve para comigo. À Doutora Daniela Machado, pela supervisão, pelos longos dias de experiências com almoços tardios, e fins-de-semana de trabalho intenso, pela amizade, paciência e preocupação.
A todo o pessoal do laboratório do CBQF que tive prazer de conhecer, pelo bom ambiente de trabalho, pelas gargalhadas de corredor, pelas small talks
entre experiências que foram capazes de amenizar os dias mais complicados. Em especial à Dina, pela amizade, pelo apoio incondicional e pelas late nights
no laboratório e à Sara e ao Eduardo, pelo acompanhamento e apoio prestados.
À Catarina, Mariana, Leandro e Feliciana que, quando cansada e desanimada, sempre me providenciaram com momentos de alegria e distração. Pelos “só mais 5 minutos ao sol por favor!”
Aos meus colegas de mestrado, pelo companheirismo que nos permitiu superar obstáculos, pelas longas viagens de comboio, pelas conversas que “não lembram a ninguém”, pelas risadas de fazer doer a barriga. Pela família criada da qual nunca me vou esquecer.
À Joana, pela presença constante desde o inicio desta caminhada. Pelo apoio, amizade. Pelo “se tu fores eu também vou”.
À Ju e ao Pedro, pela amizade de muitos “anos e troca-o-passo”, pelas sessões de trabalho, pelas conversas sem necessidade de palavras, pelo apoio sem fim que sempre demonstraram.
Aos meus amigos mais próximos, pela amizade, preocupação e incansável apoio.
À minha família, que apesar de todas as adversidades, me mostra todos os dias que na união há força, que só com amor alcançamos vitória e que o sorriso é a nossa divisa. Acima de tudo, aos meus pais e irmãos, pela paciência, encorajamento e amor manifestado. Pela compreensão demonstrada nos meus piores dias. Não há preço neste mundo que pague os sacrifícios que fizeram por mim.
palavras-chave Akkermansia muciniphila, Probióticos de Nova Geração, disbiose, formulação, microencapsulação, simulação gastrointestinal
resumo Nos últimos anos, a comunidade científica tem vindo a reunir um maior conhecimento das dinâmicas que estão na base dos distúrbios metabólicos e inflamatórios, muitos dos quais relacionados com a alimentação. O intenso crescimento destes distúrbios está a atingir proporções epidémicas, trazendo novos desafios aos clínicos e investigadores. As funções moduladoras e as propriedades específicas que as bactérias benéficas/probióticas possuem no contexto do ecossistema intestinal, parecem ser a chave para prevenir tais perturbações. Atualmente, Akkermansia muciniphila tem emergido como um “probiótico do futuro ou de nova geração" (“Next Generation Probiotics” – NGP), dado o seu potencial na prevenção e tratamento de distúrbios inflamatórios/cardio-metabólicos. Os desafios envolvendo esta bactéria probiótica residem principalmente na sua sensibilidade à atmosfera aeróbia e baixo pH. Por estas razões, esta tese tem como objetivo explorar formulações liofilizadas envolvendo agentes protetores tais como antioxidantes, prebióticos e agentes de volume bem como a microencapsulação como estratégias tecnológicas para aumentar a viabilidade da A. muciniphila face à passagem no trato gastrointestinal (GI) e promover a sua estabilidade durante o armazenamento aeróbio.
Primeiramente, uma caracterização fenotípica da estirpe A. muciniphila DSM 22959 foi efetuada. Nesta análise, características morfológicas e a coloração face à técnica de Gram, confirmam a sua natureza Gram-negativa e morfologia cocobacilar. Além disso, foi demonstrado que os ácidos miristoleico e pentadecanóico são os principais ácidos gordos presentes na membrana de A. muciniphila. Adicionalmente, as suas colónias foram caracterizadas como sendo pequenas, circulares e translúcidas.
A exposição ao ar ambiente revelou a capacidade de sobrevivência de A. muciniphila até 60 horas em atmosfera aeróbia, a 37 ºC. Apesar da tendência de declínio na viabilidade, a A. muciniphila foi capaz de sobreviver à atmosfera aeróbia durante 60 h.
Também, as propriedades de adesão desta bactéria ao epitélio intestinal foram comprovadas usando duas linhagens epiteliais, nomeadamente Caco-2 e HT29-MTX. Após caracterização fenotípica, formulações liofilizadas e um método de encapsulação foram explorados como estratégias tecnológicas para promover a viabilidade e estabilidade de A. muciniphila quando expostas ao trato GI e armazenamento aeróbio. No geral, obtiveram-se valores elevados nos liofilizados com a formulação contendo inulina (10 % m/v), riboflavina (16.5 mM) e glutationa (0.2 % m/v) do que no seu liofilizado homólogo com amido (10.2 vs 6.3 log UFC g-1). Além disso, a adição de amido à formulação conferiu maior estabilidade durante o armazenamento aeróbico. No entanto, em ambas as formulações A. muciniphila demonstrou maior suscetibilidade ao trato GI e ao armazenamento aeróbio do que na sua forma não-formulada. Numa tentativa de reduzir a sensibilidade face ao trato GI e armazenamento aeróbio, A. muciniphila foi encapsulada através do método de emulsificação/gelificação interna, numa matriz contendo alginato-Na (4 % m/v), CaCO3 (500 mM) e isolado de proteína de soro de leite desnaturado (DWPI; 10
% m/v). Akkermansia muciniphila foi eficientemente encapsulada (95.8 ± 0.01 %), em que o diâmetro das microcápsulas foi menor do que 100 µm. Para além disso, A. muciniphila encapsulada demonstrou elevada resistência às condições GI e ao armazenamento aeróbio, uma vez que a sua viabilidade apenas decresceu um ciclo logarítmico após exposição simulada ao trato GI apresentando elevada estabilidade após 7 dias de armazenamento aeróbio, a 4ºC. Em suma, as microcápsulas de alginato-Na:CaCO3:DWPI revelaram ser a
melhor estratégia na proteção de A. muciniphila contra as condições desfavoráveis do trato GI e de armazenamento em aerobiose.
keywords Akkermansia muciniphila, Next Generation Probiotics, dysbiosis, formulation, microencapsulation, gastrointestinal simulation
abstract In recent years, the scientific community has been gathering increasingly more insight on the dynamics that are at play in metabolic and inflammatory disorders many of which are diet-related. These rapidly growing conditions are reaching epidemic proportions, bringing new challenges to clinicians and researchers. The specific roles and modulating properties that beneficial/probiotic bacteria hold in the context of the gut ecosystem seem to be a key strategy to avert such imbalances. Currently, Akkermansia muciniphila
has emerged as a potential next generation probiotic (NGP) given its demonstrated potential in prevention and treatment of inflammatory/cardio-metabolic disorders. The challenges of this non-conventional native gut bacterium lie mainly on its sensitivity to aerobic environments and low pH conditions. Based on these rationales, this thesis aims to explore freeze-dried formulations involving protective agents such as antioxidants, prebiotics and bulking agents, and microencapsulation as technological strategies to increase
A. muciniphila viability throughout gastrointestinal (GI) passage and stability under aerobic storage.
Firstly, a comprehensive phenotypic characterization involving A. muciniphila
DSM 22959 strain was conducted. In this analysis well-known staining and morphological traits namely Gram-negative and coccobacillary-shape were confirmed; furthermore, myristoleic and pentadecanoic acids were demonstrated to be the major membrane fatty acids in A. muciniphila. In addition, their colonies were morphologically characterized as being small, circular and translucent. Exposure to ambient air revealed that A. muciniphila
survived up to 60 hours in an aerobic atmosphere at 37ºC. In addition, the adhesion properties of A. muciniphila to gut epithelium were proven, using Caco-2 and HT29-MTX cell lines as in vitro models. Upon phenotypic characterization, freeze-dried formulations and encapsulation methods were explored as technological strategies to enhance viability and stability of A. muciniphila when submitted to both GI transit and aerobic storage. Overall, A. muciniphila achieved high numbers in freeze-dried powders of the formulation containing inulin (10 % w/v), riboflavin (16.5 mM) and glutathione (0.2 % w/v). In addition, this formulation matrix contained higher number of viable cells than the starch counterpart (10.2 vs 6.3 log CFU g-1), yet the addition of starch to the formulation conferred higher stability during aerobic storage. Nevertheless, in both freeze-dried formulations A. muciniphila displayed a higher susceptibility to GI transit and aerobic storage than non-formulated cells.
In an attempt to reduce sensitivity to GI and aerobic storage conditions, A. muciniphila was encapsulated, by emulsification/internal gelation method, in a Na-alginate (4 % w/v), calcium carbonate (CaCO3; 500 mM) and denatured
whey protein isolate (DWPI; 10 % w/v) matrix. Akkermansia muciniphila was efficiently encapsulated (95.8 ± 0.01 %) via such microencapsulation method, where microcapsules size diameter was smaller than 100 µm. Moreover, encapsulated A. muciniphila demonstrated high resistance to GI conditions and aerobic storage since their viability only decreased 1 log cycle after simulated GI tract exposure presenting a high stability after 7 days of refrigerated aerobic storage.
In conclusion, Na-alginate:CaCO3:DWPI microcapsules reveal a better strategy
to protect A. muciniphila against detrimental gastrointestinal transit and aerobic storage conditions.
Table of Contents
List of Abbreviations ... i
Figure Index ... ii
Table Index ... iii
Scientific outputs ... iv
1. Introduction ... 1
1.1. Probiotics: a new game plan ... 2
1.2. NGP: Akkermansia muciniphila – A new contender ... 3
1.2.1. Akkermansia muciniphila in disease ... 5
1.2.2. Akkermansia muciniphila: a peacekeeper ... 8
1.2.3. Akkermansia muciniphila: friend or foe? ... 9
1.2.4. Akkermansia muciniphila: modulation by diet... 10
1.3. Strategies for new probiotic carriers ... 12
1.4. Thesis aim ... 14
2. Material and Methods ... 16
2.1. Phenotypic characterization of A. muciniphila DSM22959 ... 16
2.1.1. Akkermansia muciniphila cultivation under reference conditions ... 16
2.1.2. Growth kinetics and cellular characterization ... 17
2.1.3. Oxygen tolerance ... 18
2.1.4. Adhesion assays to intestinal epithelium cells ... 18
2.1.5. Cell membrane lipid extraction and fatty acids methyl esters (FAME) analysis……. ... …….19
2.1.6. EOS bacterial enumeration using Flow Cytometry ... 20
2.2. Design and study of technological strategies ... 21
2.2.1. Design and preparation of freeze-dried formulations incorporating A. muciniphila ... 21
2.2.2. Establishment of novel microencapsulation strategy to increase the viability/stability of EOS and microaerophilic bacteria ... 24
2.3. Statistical analysis ... 28
3. Results & Discussion ... 29
3.1. Phenotypic characterization of A. muciniphila DSM22959 ... 29
3.1.1. Growth Curve and cell/colony morphology ... 29
3.1.3. Akkermansia muciniphila capacity to bind to human intestinal epithelial
cells………...34
3.1.4. Cell membrane fatty acids of Akkermansia muciniphila DSM22959 ... 38
3.1.5. Flow Cytometry (FCM) as a fast and simple technique for evaluation of viability of EOS bacteria ... 39
3.2. Design and study of technological strategies ... 43
3.2.1.1. Effect of prebiotics on growth of A. muciniphila ... 43
3.2.1.2. Effect of antioxidant and/or redox agents in freeze-dried formulations incorporating A. muciniphila ... 45
3.2.1.3. Impact of different formulations on A. muciniphila survival and resistance to GI………...46
3.2.1.4. Stability of the formulated A. muciniphila during storage ... 50
3.2.2. Microencapsulation ... 53
3.2.2.1. Encapsulation Efficiency ... 54
3.2.2.2. Microcapsule morphology and size ... 55
3.2.2.3. Stability of the microencapsulated A. muciniphila during storage ... 57
3.2.2.4. Survival rate of free and microencapsulated A. muciniphila exposed to simulated gastrointestinal conditions over storage ... 58
4. Concluding Remarks ... 61
5. Future Work ... 63
List of Abbreviations
2-OG 2-oleoylglycerol
ANOVA Analysis of variance
CaCl2 Calcium chloride
Caco-2 Caucasian colon adenocarcinoma
CaCO3 Calcium Carbonate
CFU Colony-forming units
DMEM Dulbecco’s Modified Eagle’s Medium
DMSO Dimethyl sulfoxide
DWPI Denatured WPI
EE Encapsulation efficiency
FA Fatty acid
FAME Fatty acids methyl esters
FBS Fetal Bovine Serum
FCM Flow Cytometry
FISH Fluorescent in situ hybridization
FOS Fructo-oligosaccharide
FSC Forward-scatter
GI Gastrointestinal
GLP-1 Glucagon-like peptide-1
GP Gastric Phase
HT29-MTX Mucus secreting human colon adenocarcinoma
IFN-γ Interferon gamma
IP Intestinal phase
LAB Lactic acid bacteria
LPS Lipopolysaccharides
MAMP Microbial associated-molecular pattern
MUFA Monounsaturated fatty acids
Na-alginate Sodium alginate
NGP Next-Generation Probiotic
OD Optical density
OP Oral phase
PB Phosphate buffer
PBS Phosphate buffer saline
PI Propidium iodide
Rpm Revolutions per minute
rt-PCR Real-time quantitative polymerase chain reactions
SCFA Short-chain fatty acids
SFA Saturated fatty acids
SGF Simulated gastric fluid
SIF Simulated intestinal fluid
SSC Side-scatter
SSF Simulated salivary fluid
T2D Type 2 diabetes mellitus
TO Thiazole orange
VBNC Viable but not culturable
W/O Water-in-Oil
Figure Index
Figure 1.1 - Graphical summary of Akkermansia muciniphila interactions in gut epithelium responsible for maintaining host health.. ... 4 Figure 1.2 - Schematic flow chart of thesis outline. ...15 Figure 2.1 - Schematic representation of in vitro GI procedure based on Minekus et al.(2014) with some modifications. ...23 Figure 2.2 - Schematic overview of microencapsulation of Akkermansia muciniphila by emulsification/internal gelation method. ...26 Figure 3.1 - Growth curve of Akkermansia muciniphila DSM22959. ...30 Figure 3.2 - Photos of Akkermansia muciniphila DSM 22959 CFUs in PYGM agar after 7 d of incubation at 37 ºC under anaerobic conditions. ...30 Figure 3.3 - Cells of Akkermansia muciniphila DSM22959 after Gram stain visualized under optical microscope (100 x). ...31 Figure 3.4 - Variation of viable cells (log CFU mL-1) of A. muciniphila in PYGM broth exposed to aerobic conditions (ambient air) without agitation (■) or under agitation (▲) and to anaerobic conditions (●), at 37 ºC. ...32 Figure 3.5. Viable cells of A. muciniphila in the adhesion inoculum (Initial) and resulting of the adhesion to Caco-2 (dark blue) or HT29-MTX (light blue) cell lines throughout the short adhesion time assay. ...35 Figure 3.6 - Viable cells of A. muciniphila adhesion inoculum (initial) and resulting of the adhesion to Caco-2 cell line by A. muciniphila DSM 22959 throughout the long-term adhesion assay. Different letters represent the statistically significant differences (p < 0.05) found between sampling times. ...36 Figure 3.7 – Flow cytometric analysis of A. muciniphila DSM22959. Data acquisition was obtained with a single dye (TO). ...41 Figure 3.8 - Values of OD at 600 nm (a) and of viable cell numbers [Log (CFU mL-1)] (b) of A. muciniphila grown in PYGM broth with or without prebiotic agents [I: Inulin 2.5% (w/v); FOS: FOS 2.5% (w/v)].. ...44 Figure 3.9 - Freeze-dried formulations incorporating A. muciniphila: I+R, formulation with 10 % (w/v) inulin and 16.5 mM riboflavin;. ...45 Figure 3.10 - Viable cell numbers of A. muciniphila in PYGM broth ( ) [Log (CFU/mL)], incorporated in dried core formulation (■) [Log (CFU/g)] and incorporated in freeze-fried in core formulation with starch (▲) [Log (CFU/g)] during simulated gastrointestinal conditions ...47 Figure 3.11. Morphology of wet microencapsulates, as assessed under inverted optical microscope post-microencapsulation (0 d). ...56 Figure 3.12. Viable cell numbers of free (●) [Log (CFU/mL)] and microencapsulated (■) [Log (CFU/g)] A. muciniphila during 14 days of aerobic storage at 4ºC. ...57 Figure 3.13 - Viable cell numbers of free [Log (CFU mL-1)] and microencapsulated [Log (CFU g-1)] A. muciniphila during simulated GI conditions, after aerobic storage, at 4 ºC. .59
Table Index
Table 2.1 - Composition of Akkermansia muciniphila reference growth medium (PYGM broth) according to DSMZ. ...17 Table 2.2 - Composition of salt solution included in Akkermansia muciniphila reference growth medium according to DSMZ. ...17 Table 2.3 - Composition of electrolyte stock solutions for each phase of the GI protocol according to Minekus et.al. (2014). ...22 Table 3.1 - Percentage values of relative adhesion of A. muciniphila to Caco-2 and HT29-MTX cell lines for the short-term assay. ...35 Table 3.2 - Percentage values of relative adhesion of A. muciniphila to Caco-2 cell line for the long-term assay. ...36 Table 3.3 – Fatty acid composition (of A. muciniphila DSM22959 cell membrane after 17 ± 2h incubation in PYGM broth. ...38 Table 3.4 – Freeze-dried mass (mean ± SD; g) of formulation with or without starch incorporating A. muciniphila. ...51 Table 3.5 - Viability of A. muciniphila DSM2295 cells in PYGM (log CFU mL-1) and formulated (with/without starch) (log CFU g-1) stored aerobically at 4 ºC and -20 ºC for 7 days. ...52 Table 3.6 - Viability (log CFU g-1) A. muciniphila DSM22959 cells, formulated with and without starch, stored aerobically at ambient air (22 ºC) for 4 days. ...53
Scientific outputs
Paper in Peer Reviewed Journals
Almeida D. et al. Submitted. Evolving trends in next-generation probiotics: a 5W1H perspective. Critical Reviews in Food Science and Technology. Submitted at 3rd August. BFSN-2018-3570.
Poster Presentations
Freitas A., Almeida D. et al. 2018. Strategies to increase Akkermansia muciniphila viability during simulated gastrointestinal conditions and stability storage. Poster presented in the 6th World Congress on Targeting Microbiota, Porto, 28-30th October. Abstract in page 75, Book of Abstracts.
Freitas A. Almeida D. et al. 2018. Formulation strategies for enhancing growth of Akkermansia muciniphila and its survival through lyophilisation and storage at air ambient. Poster presented in the 12th International Scientific Conference on Probiotics, Prebiotics, Gut Microbiota and Health – IPC2018, Budapeste, 18-21th June. Abstract in page 71, Book of Abstracts.
1.
Introduction
Worldwide, type 2 diabetes mellitus (T2D), obesity and inflammatory bowel diseases (IBD) among others have achieved high proportions, constituting serious public health problems with important social, financial and health systems implications (GBD 2015 Obesity Collaborators et al. 2017; Ogurtsova et al. 2017; Ng et al. 2018) Furthermore, these metabolic and inflammatory conditions have been related with dramatic changes in the human gut microbiota at both quantity and quality (bacterial species diversity) levels. Indeed, the gut microbiota associated with the gut epithelial barrier plays a key role in the regulation of the inflammatory and metabolic host profiles, promoting an overall host cellular homeostasis status contributing to global health and minimizing the triggering of inflammatory mechanisms (Hartstra et al. 2015; Marchesi et al. 2016; Cani 2017). Recently, de Vos and colleagues (2012), described the nature of the relationships between the microbiota profile and associated intestinal diseases suggesting that the intestinal microbiome could be linked to a growing number of over 25 diseases or syndromes (De-Vos & De-Vos 2012).
It is still not clear how the interaction between microbiome and host immunity affects the development of specific diseases, yet it is established that exposure to low bacterial diversity in early life can prevent or delay immune mucosa maturation reducing immunological tolerance and hence increasing the risk of aberrant immune response and allergic disease (Brooks et al. 2013). In addition to the immune modulating properties, the gut microbiota also contributes to the host condition by granting a protective barrier against pathogens, enabling digestion through the breakdown of non-digestible food constituents and producing essential metabolites (Ottman et al. 2012). Since low microbial diversity has been associated with several life-style related non-communicable diseases such as obesity, metabolic syndrome, immune-related, and inflammatory diseases (D’Argenio & Salvatore 2015), a microbial ecosystem with higher diversity can be considered as an indicator of a more healthy status (Jordán et al. 2015), as demonstrated in elderly subjects (Jeffery et al. 2016).
One of the most attainable approaches for modulation of gut microbiota diversity is through dietary interventions. Studies focusing on the negative impact of westernization diet have demonstrated the co-evolution of microbial species and the human host (Cordain et al. 2005; Blaser & Falkow 2009; Quercia et al. 2014). It is important to consider that dietary intake, dependent on its nature and quantity, may promote imbalances in colonic microbial populations leading to the onset of numerous inflammatory conditions that may degenerate into chronic diseases (Celiberto et al. 2015). In addition to certain well-balanced diets, such as the Mediterranean diet
(Garcia-Mantrana et al. 2018), specific food choices such as high-fat dairy products (Bordoni et al. 2017) or dietary fiber (Masuko 2018), associated to pro- (in subjects with milk allergy) and anti-inflammatory effects, respectively, can significantly modulate gut microbiota composition. Furthermore, this relationship is more dynamic than initially thought, as the active “adjustment” of microbial composition - e.g. probiotic and synbiotic supplementation (Plaza-Díaz et al. 2017) - can also elicit anti-inflammatory effects. Given these recent findings, overcoming host genetic predisposition for the development of inflammatory and metabolic conditions through dietary interventions or more actively, via new generation probiotics supplementation, may be the future strategy choice for therapy or, even as a prevention tool to avoid the onset of these chronic diseases, fueling hope for the millions of patients worldwide that experience the taxing consequences of inflammatory/metabolic conditions.
1.1. Probiotics: a new game plan
Upholding the above considerations, new actions are required for the prevention and treatment of such impacting inflammatory diseases. In recent years, several studies have demonstrated that the consumption of selected microbes, named probiotics, are a highly promising therapeutic alternative for treating the gut microbiota dysbiosis (Cani & Van Hul 2015). In 1908, Ellie Metchnikoff introduced the concept of probiotics stating that "the dependence of the intestinal microbes on food makes it possible to adopt measures to modify the flora in our bodies and to replace the harmful microbes by useful microbes" (Metchnikoff 1908). Currently, the most sound-based definition accepted worldwide for probiotics is that they are “live microorganisms that, when administered in sufficient quantities, confer a health benefit on the host” (Hill et al. 2014). In fact, probiotics inhibit pathogen colonization by competitive exclusion and displacement, enhance intestinal barrier function and modulate the immune and neuroendocrine systems, locally and systemically, as evidenced in in-vitro and animal trials (Neef & Sanz 2013).
Conventionally, the microorganisms belonging to the Lactobacillus and Bifidobacterium genera have been employed as probiotics, encountering wide commercial availability in the market (Douillard & de Vos 2014). These microorganisms are usually well-accepted and tolerated by humans (Brodmann et al. 2017). However, these classical probiotics display limited effects on the human gut microbiota, thus calling for a better selection of bacterial strains and formulation of delivery vehicles (Neef & Sanz 2013). In this context, several bacterial species comprising genera other than Lactobacillus and
Bifidobacterium with proven efficacy have been considered as potential next-generation probiotics (NGPs), namely strains from the Akkermansia, Eubacterium, and Faecalibacterium genera (Cani & Van Hul 2015; Patel & DuPont 2015; O’Toole et al. 2017; O’Toole & Paoli 2017).
1.2. NGP: Akkermansia muciniphila – A new contender
Among the more recent proposals of new species as NGPs, Akkermansia muciniphila stands out as an interesting candidate for this category which should possess high potential to avert inflammatory and diet-related disorders (Gómez-Gallego et al. 2016; Cani 2018; Naito et al. 2018). Akkermansia muciniphila belongs to the phylum Verrucomicrobia and is a common resident of the human gut, representing approximately 1-3 % of the total gut microbiota (Derrien et al. 2008). Its cells are oval-shaped, non-motile and stain Gram-negative (Derrien et al. 2004). Akkermansia muciniphila was originally classified as a strict anaerobe (Derrien et al. 2004), but recently it was demonstrated that itcan use oxygen in nanomolar concentrations, being reclassified as an aerotolerant anaerobe (Ouwerkerk et al. 2016).
The particular feature of this bacterial species is the ability to degrade mucin, a high molecular mass glycoprotein, known as the main component of the intestinal mucus and an important mediator of the gut barrier (Derrien et al. 2004; Derrien et al. 2017). The colonic mucus coating the epithelial layer of the gastrointestinal (GI) tract (Figure 1.1), produced by goblet cells, serves as the primary barrier of the colon protecting the host against toxins and pathogens and its thickness reduction increases gut permeability and the risk of endotoxemia as well as bacterial translocation (Johansson et al. 2014). Additionally, the A. muciniphila unique ability to utilize mucin as a stable sole source of nitrogen and carbon (Derrien et al. 2004) also provides this microorganism with an ecological advantage as in the case of glycan deficiency (Derrien et al. 2017).
Figure 1.1 - Graphical summary of Akkermansia muciniphila interactions in gut epithelium responsible for maintaining host health. Colonic mucus, produced by goblet cells, is degraded by A. muciniphila leading to the production of bioactive molecules, such as acetate and propionate (SCFAs), and the release of oligosaccharides utilized by non-mucus degrading butyrate – producing bacteria (Anaerostipes caccae, Eubacterium hallii and Faecalibacterium prausnitzii) for butyrate production. Butyrate is linked to distinct beneficial effects on the host improving the intestinal barrier integrity which prevents metabolic endotoxemia (uptake of LPS) a key characteristic of metabolic disorders such as type 2 diabetes and insulin resistance; it also exerts immunoregulatory properties promoting an anti-inflammatory phenotype. Glucose tolerance can be mediated by A. muciniphila through the control of the negative effects of IFNγ and, also through the increase of gut hormone production such as GLP-1 via production of 2-OG (endocannabinoid system). In addition, A. muciniphila induces the production of the antimicrobial peptide RegIIIγ via Paneth Cell synergistically increasing the anti-inflammatory milieu of the gut ecosystem.
Abbreviations: Treg cells, regulatory T cells; Anti TH, Anti-inflammatory cytokines producing T cells; Pro TH, Pro-inflammatory cytokines producing T cells; GPCR, G protein–coupled receptor; GLP1/2, Glucagon-like peptide-1 and 2; IFNγ, Interferon gamma; RegIIIγ, Regenerating islet-derived 3 gamma; 2-OG, 2-oleoylglycerol.
The specialized mucin fermentation results in the production of short-chain fatty acids (SCFAs) such as acetate and propionate (Derrien et al. 2004), as may be seen in Figure 1.1. This phenomenon may also influence gut bacterial composition, by creating a favorable environment for the growth of strict anaerobes, in a syntrophic association, such as non-mucus degrading butyrate-producing bacteria, which could have a synergistic effect on the host (Rios-Covian et al. 2015; Belzer et al. 2017). Moreover, A. muciniphila partakes on colonic mucus turnover cycle since the produced SCFAs also stimulate goblet cells mucus production (Shimotoyodome et al. 2000). Hence, A. muciniphila cell proportion is correlated with an increase of the number of the gut mucus-producing goblet cells, thus sustaining the intestinal barrier integrity (Shin et al. 2014). Indeed, an in vitro model demonstrated that A. muciniphila adheres to the gut epithelial cells, fortifying enterocyte monolayer integrity (Reunanen et al. 2015; Chelakkot et al. 2018).In addition to its effect on gut barrier function, A. muciniphila is linked to a desirable metabolic profile and to the reduction of fat mass without interfering with total energy intake by the host (Everard et al. 2013; Dao et al. 2016). These host-microbe-overall metabolism interactions are probably mediated by A. muciniphila endocannabinoid system since it substantially increases intestinal levels of 2-oleoylglycerol (2-OG), a bioactive lipid involved in gut hormone synthesis and appetite control (Figure 1; Table 1).
1.2.1. Akkermansia muciniphila in disease
Recently, it has been reported that A. muciniphila can be used as a biomarker of a healthy host metabolic profile. In fact, a reduction of A. muciniphila levelsin the gut has been related with several metabolic and inflammatory diseases, such as obesity, T2D and IBD (Png et al. 2010; Cani & Everard 2014; Schneeberger et al. 2015). Recent mechanistic studies related with A. muciniphila provide insight on what type of physiological routes are at play for the favourable anti-inflammatory and antimicrobial attributes this bacterium offers the host for protection against disease(Table 1).
Unquestionably, and as previously mentioned, one of the most relevant mechanisms for A. muciniphila probiotic action is the strengthening of the gut epithelial barrier against the diffusion of potentially immune-activating substances - such as bacteria, endotoxins, and digestion products - from the gut content that, when in contact with intestinal tract immune system, trigger the onset of systemic inflammation (Cani et al. 2007). This process, also termed as metabolic endotoxicity, is due to the damage of the gut barrier integrity such as the breakdown of the intercellular junction between the intestinal mucosal walls, which causes an alteration in intestinal permeability, a
condition known as leaky gut (Michielan & D’Incà 2015). Low grade inflammation is recognized to be one of the triggers involved in the metabolic shifts that occur upon cardio-metabolic diseases such as obesity and T2D (Gomes et al. 2017; Cani et al. 2007). Indeed, it has been demonstrated that the increased levels of circulating lipopolysaccharides (LPS), a component of Gram-negative bacteria, is one of the main factors causative of these conditions (Cani et al. 2007; Reunanen et al. 2015).
An increase in A. muciniphila numbers is also linked to an improved adipose tissue metabolism increasing the endogenous production of specific bioactive lipids such as 2-OG. This bioactive lipid stimulates the secretion of glucagon-like peptides, such as glucagon-like peptide-1 (GLP-1) (Everard et al. 2013), and its release results in an interaction with the host endocrine system which in turn influences gut barrier function and energy homeostasis (Everard et al. 2011). More recently, a specific bacterial structural component protein Amuc_1100 found on the outer membrane of A. muciniphila and implicated in the formation of pili (Plovier et al. 2016) has been shown to have an important immunomodulatory action in both in vitro and in vivo models (Ottman et al. 2017; Cani 2018). Indeed, Amuc_1100 protein seems to be involved in the reduction of fat mass development and dyslipidemia, as well as in insulin tolerance improvement (Plovier et al. 2016).
Another important biological property is related to its ability to improve glucose tolerance. In this context, Greer and colleagues showed that A. muciniphila can mediate the negative effects of interferon gamma (IFN-γ) on glucose tolerance (Greer et al. 2016).
Table 1 - Summary of beneficial impacts on human health and disease by the NGP Akkermansia muciniphila
Target Conditions Key Finding(s) Study Type Refs
Inflammation
Increases enterocyte monolayer integrity by promoting colonic mucus turnover and lowering LPS uptake In vitro (adhesion assay)
(Reunanen et al. 2015; Derrien et al. 2017) Amuc_1100 (outer membrane protein implicated in the formation of pili) can recapitulate the beneficial
effects of the whole live bacterium; shows stability at temperatures used in pasteurization (via the specific activation of Toll-like receptor 2)
In vitro (Plovier et al. 2016;
Ottman et al. 2017)
Stimulates RegIIIγ (antimicrobial peptide against Gram-positives) production by Paneth cells Preclinical in mice (ob/ob mice model)
(Everard et al. 2013); (Pott & Hornef 2012).
Metabolic disorders (diabetes, obesity)
Negatively correlated with intestinal permeability, metabolic endotoxaemia, inflammatory biomarkers and low grade induced metabolic disorders such as T2D and insulin resistance with additional increased macrophage infiltration into the adipose tissue and hepatic steatosis
Case control studies
(Cani & Van Hul 2015; Dao et al. 2016) Mediates IFN-γ (pro-inflammatory cytokine, responsible for the control of intracellular pathogenic infections)
adverse effects on glucose metabolism;
Abundance negatively controlled by Irgm1 gene (controls A. muciniphila levels)
Preclinical in mice
(IFN- γ KO models) (Greer et al. 2016) Prebiotic administration reduced adiposity, inflammatory markers, insulin resistance, and improved gut
barrier (via T regulatory cell induction in adipose tissue and NOD-like receptor pyrin domain containing 6 - NLRP6 - inflammasome assembly)
Preclinical in mice
(Everard et al. 2013; Schneeberger et al. 2015; Anhê et al. 2016)
Negatively correlated with insulin intolerance Case control
studies (Zhang et al. 2013)
Improves metabolic profile and reduced fat mass (increases 2-OG, a bioactive lipid that stimulates the secretion of glucagon-like peptides through the activation of GPR119, via the endocannabinoid system)
Case control study
Preclinical in mice (Dao et al. 2016) Higher abundance of A. muciniphila-derived extracellular vesicles (AmEVs) in healthy control vs T2D
patients.
Reduced gut permeability in LPS-treated Caco-2 cells with AmEVs treatment.
Preclinical in mice (Chelakkot et al. 2018)
Atherosclerosis
Ameliorates metabolic endotoxaemia-induced inflammation (via restoration of the gut barrier endotoxaemia);
Reduction of the expression chemokines and the adhesion molecules MCP-1, TNFα, and ICAM-1, along with decreased aortic infiltration of macrophages
Preclinical in mice
1.2.2. Akkermansia muciniphila: a peacekeeper
In the framework of gut health, the identification of microbiota community members and, more importantly, their role is crucial to better understand the dynamics that are at play to maintain such an ecosystem stable and healthy (Trosvik & de Muinck 2015). One undervalued concept in biology is that of keystone species, initially defined as a species critical for managing the diversity and organization of their ecological communities through biotic interactions with other community members (Paine 1969; Trosvik & de Muinck 2015). The distinct aspect of a keystone species is that considering its relatively low abundance; it has a disproportionately substantial effect on the community. Thus its removal has strong destabilizing impact, resulting in loss of biodiversity which represents a vulnerable point in an ecosystem (Stachowicz & Hay 1999). It should also be noted that a microorganism can even be considered as a keystone member if pivotal to an ecosystem by producing essential metabolites such as SCFAs that set off trophic cascades, strengthening its defenses against pathogenic species, aiding the establishment of beneficial species, and overall helping to preserve a balanced relationship with the host (Laforest-Lapointe & Arrieta 2017). Indeed, research suggests that the abundance of certain keystone species are responsible for the individuality of the human gut microbiome (Fisher & Mehta 2014). Even though this concept may seem not to entirely apply to A. muciniphila regarding its relative abundance, it provides a proper context to understand the reasons why these microorganisms are viewed as peacekeeping players and prime targets for maintenance of intestinal health through manipulation of the GI microbiota (Trosvik & de Muinck 2015).
Among keystone species commonly present in gut microbiota, A. muciniphila has been highlightned as a potential entrance point for novel diagnostic strategies and therapeutic modulation (El Hage et al. 2017) not only due to its bioactive properties but also due to interesting microbial cross-feeding dynamics and symbiotic relationships that occur at the intestinal mucus layer that support other species growth and maintaining the gut microbiome functioning as a whole unit (De Vuyst & Leroy 2011; Belzer et al. 2017). Cross feeding is a valuable microbial feature, where metabolic products produced, for example from the metabolism of dietary prebiotics by one species serve as a substrate for other species, allowing the retrieval of nutrients and energy through fermentation (Chassard & Lacroix 2013).
As a mucolytic microorganism A. muciniphila supplies sugars, via the degradation of complex glycans such as mucin, to butyrate-producing bacteria like Eubacterium hallii and Anaerostipes caccae (Belenguer et al. 2006; Schwab et al. 2017). Additionally, A. muciniphila mucin degradation activity, which also increases acetate and propionate
pool, can also benefit the butyrate-producing Faecalibacterium prausnitzii (Rios-Covian et al. 2015). Butyrate has several beneficial effects on the host, including the provision of an energy source for epithelial cells, induction of colonic regulatory T cells, induction of apoptosis in human colonic carcinoma cells, inhibition of inflammatory responses in intestinal biopsy specimens and improvement of metabolic syndrome (Reigstad et al. 2015; Yano et al. 2015; Brodmann et al. 2017).Furthermore, a vitamin B12-dependent syntrophy between E. hallii and A. muciniphila was observed, providing A. muciniphila a necessary cofactor for the production of propionate, benefiting host cell metabolism (Belzer et al. 2017). Overall, cross-feeding interactions evidence broadens our understanding in that modulation of a gut keystone species, such as A. muciniphila, impacts dramatically the intestinal microbial ecosystem and the associated host-microbiota equilibrium.
1.2.3. Akkermansia muciniphila: friend or foe?
In some cases, the mechanism by which this bacteria aids in the amelioration of metabolism and protects LPS endotoxaemia, namely the mucus turnover cycle (Derrien et al. 2004), can also contribute to the reduction of the gut barrier integrity and function (Sonoyama et al. 2010). Studies proposed that this action can lead to an increase in the uptake of allergenic proteins, such as ovalbumin, in the GI tract (Sonoyama et al. 2010). It is important to note, however, thatno evidence was shown supporting that A. muciniphila alone imparts pathogenic characteristics. Comparatively, Zheng et al. (2016) demonstrated that the higher amounts of A. muciniphila and of pathogenic bacteria belonging to Enterococcus and Shigella genera, alongside with the decreased abundance of bacterial groups with anti-inflammatory action (e.g. Bacteroides fragilis and Streptococcus salivarius), may contribute to eczema in infants (Zheng et al. 2016). Moreover, the collapse of the gut barrier integrity can also lead to increased levels of inflammatory microbial associated-molecular patterns (MAMPs) in the blood circulation potentially contributing to neuro-inflammation (Derrien et al. 2017). Additionally, A.muciniphila was also positively correlated with colorectal cancer patients, with about 4-fold higher numbers versus the healthy subjects involved in the study (Weir et al. 2013). It is, however, important to mention that patients with cancer usually have decreased food intake, and studies showcase that fasting correlates with increased A. muciniphila levels (Remely et al. 2015). Furthermore, this type of cancer is related to increased mucus production and cell proliferation, which in turn can potentiate the boost in this mucus degrading bacterium abundance (Gómez-Gallego et al. 2016). Thus, it appears that themucin degrading role of A. muciniphila, which can
be viewed as a typical pathogen-like behavior, is in fact regarded as a regular process in a self-renewing healthy intestine (Gómez-Gallego et al. 2016). Ultimately, even though the possible downside of its physiological behaviors still needs to be more addressed, the overwhelming current evidence correlating A. muciniphila and human health offers the possibility of not only using it as a near future therapeutic approach but also as a diagnostic and prognostic tool for the host’s cardio-metabolic conditions.
1.2.4. Akkermansia muciniphila: modulation by diet
As previously mentioned, gut microbiota modulation can be achieved through diet. Based on its physiological influence on the host, A. muciniphila has been suggested as a prognostic tool to anticipate the success of dietary interventions. Dao et al. (2016) assessed several clinical parameters as well as A. muciniphila abundance before and after a 6-week calorie restriction period, followed by a stabilization diet. According to these researchers, it was shown that the higher abundance of A. muciniphila at baseline was linked with improvement in blood glucose homeostasis, lipid profile and body fat distribution after dietary intervention (Dao et al. 2016).
In the same way, the consumption of specific prebiotic compounds, defined as non-digestible food ingredients that pass through the upper gut and which are selectively fermented by colonic bacteria, can positively influence health through several mechanisms (Sarao & Arora 2017). Initially, it was demonstrated that prebiotic feeding resulted in improved metabolic syndrome associated conditions such as insulin sensitivity (increased GLP-1) and glucose homeostasis (Kok et al. 1998). These improvements were linked with a modulation of the gut microbiome composition and activity , mostly referring to Bifidobacterium spp. and Lactobacillus spp increased numbers and activities (Kapiki et al. 2007) but currently these benefits have been extended to other bacterial genera such as Akkermansia, Eubacterium, and Faecalibacterium (Deehan et al. 2017).
Several studies demonstrated that A. muciniphila is able to ferment human milk oligosaccharides mainly due to the chemical similarity of these glycans with mucin structure (Collado et al. 2012; Ward et al. 2013; Petschacher & Nidetzky 2016) Furthermore, this bacterium is capable of foraging specific dietary components including prebiotic compounds (Everard et al. 2011) and polyphenols (Roopchand et al. 2015; Anhê et al. 2016) to provide continuous benefits to the host. Among the many prebiotics, dietary fructan inulin and its breakdown product fructo-oligosaccharide (FOS) are particularly well-studied, and evidence supporting their health-promoting
effects, specifically their influence on host gut microbiota is accumulating quickly (Sarao & Arora 2017).
It has been demonstrated that inulin promotes A. muciniphila growth, while improving metabolic disorders associated with obesity, including reduction of fat mass and insulin resistance, lower liver steatosis and the gut barrier reinforcement (Van-den-Abbeele et al. 2011; Everard et al. 2011; Cani & Everard 2014; Greer et al. 2016) However, the specific modulation effect of inulin on A. muciniphila’s abundance appears to be due to an indirect side effect of its activity and not because of its direct use (Van Herreweghen et al. 2017). Inulin fermentation by gut resident strains lowers the pH (Van-den-Abbeele et al. 2011) which, in turn stimulates colonic mucus production (Ten Bruggencate et al. 2004); this relationship is based on the observation that colonization correlates with pH and mucin content, but not with inulin concentration (Van Herreweghen et al. 2017). Thus, it is fair to state that by increasing mucin secretion and maintaining pH homeostasis, inulin consequently promotes the growth of A. muciniphila.
FOS is another commonly studied prebiotic that seems to possess similar A. muciniphila-promoting activities. In recent in vivo studies, supplementation with FOS was found to have a significant prebiotic effect, in particular, demonstrating an increase of A. muciniphila abundance and, this increase was accompanied by fat mass reduction and improved glucose control (Everard et al. 2011; Everard et al. 2013; Burokas et al. 2017). However, FOS mechanistic modulation upon the ecology of A. muciniphila is still not well understood (Zhou 2017).
Dietary polyphenols are other bioactive compounds that seem to exert prebiotic-like effects on A. muciniphila. As natural antioxidants and antimicrobials compounds, polyphenols may create a selective pressure on the intestinal lumen by scavenging free oxygen radicals, favoring anaerobic species like A. muciniphila (Anhê et al. 2016; Daglia 2012; Roopchand et al. 2015). Although there is some evidence that dietary polyphenols do affect positively this bacterium abundance, inconsistent results suggest that the A. muciniphila-promoting effects are dependent on polyphenols chemical nature and sources (Roopchand et al. 2015; Li et al. 2015; Anhê et al. 2016)..
Overall a more systematic analysis of which specific prebiotics confer positive impact on A. muciniphila growth and stability and, by which mechanisms such modulations occur, should be investigated, since it seems to be a more passive or indirect approach. In particular, changes in diet through the introduction of compounds that possess mucin-like structures or prebiotic substrates, as inulin-type fructans and FOS, that stimulate mucus secretion in the gut, seem to provide a stable colonization niche (Moens et al. 2016), which translates into a passive strategy to be implemented in
future dietary approaches or even in probiotic technological applications, such as the inclusion in formulation matrices to support cell viability and stability.
1.3. Strategies for new probiotic carriers
Since modulation of the gut microbiota has demonstrated promising outcomes in the treatment and prevention of several disorders, introducing potential NGPs, such as A. muciniphila, in the pharmaceutical and food nutraceutical markets seems an interesting strategy for the prevention and treatment of such dysbiosis-associated diseases (O’Toole et al. 2017). However, in contrast to the fairly oxygen-tolerant probiotics that are currently commercialized, A. muciniphila´s high sensitivity to oxygen, requires new and adequate approaches to standardized experimental protocols limiting strain to ambient oxygen exposure.
To ensure oxygen exclusion from processes such as formulation and freeze-drying, strategies using the incorporation of antioxidants for redox potential reduction (Sousa et al. 2012) or the physical protection of the strain by means of encapsulation (Sousa et al. 2015) have been evidenced as useful efforts to promote probiotic viability as well as functional stability (O’Toole & Paoli 2017). The incorporation of protective agents, such as prebiotics, into bacterial formulations appears to positively impact bacterial viability, especially in commonly used processes such as freeze-drying and storage (Sarao & Arora 2017). To date, the most widely used protective agents showing satisfactory impact on cell viability and stability, include: inulin (Mensink et al. 2015), maltodextrin (Fernandes et al. 2014) and trehalose (Fowler & Toner 2005). Bacteria with particular oxygen sensitivity can also be protected against oxygen toxicity during storage, by the addition of oxygen scavengers (e.g., cysteine, glutathione, and ascorbate)(Ross et al. 2005) and through co-culture with oxygen-consuming species such as Bifidobacterium spp, Streptococcus thermophilus and yeasts (Xie et al. 2012; Ma et al. 2015), reducing oxygen content in the formulation matrix,thereby improving probiotic viability.
Another approach considered to enhance microbial robustness against not only oxygen but other factors such as pH and temperature, is the application of sub-lethal stress treatments which allow the activation of metabolic pathways that permit the re-adaptation to the aforementioned elements, in addition to other processes (freeze-drying and storage), improving the survival during industrial formulation and ultimately in the gut (Borges et al. 2012; Nguyen et al. 2016).
The viability and stability of bacterial cells can be further improved through encapsulation (Šipailienė & Petraitytė 2018). Besides offering a physical barrier against the external environment (storage temperatures and oxygen content), encapsulation also provides a controlled release into the gut lumen, which is particularly important
when it comes to ensuring survival during the passage through the GI system, mainly due to pH values (Martín et al. 2015; Anal & Singh 2007). One of the most widely used probiotic encapsulation agents is alginate, which has been linked to an increase in bacterial survival of up to 95%, depending on the species and strain (Mandal et al. 2006; Lopes et al. 2017). Alginate is a natural occurring heteropolysaccharide, comprised ofβ-D-mannuronic acid and α-L-guluronic acid (Gacesa 1988), widely used in encapsulation methods due to its low immunogenicity and good biocompatibility (Fang et al. 2007) and its resistance to pH (3 to 7) and enzymatic degradation (Klayraung et al. 2009). Within the various reported encapsulation methods, spray drying (Rodrigues et al. 2011), extrusion/external gelation (Rodrigues et al. 2012) and emulsification by internal gelation (Holkem et al. 2016) are the most well documented. Choice of the most favorable probiotic encapsulation methodology should evaluate the materials and conditions to be used before application. Despite the low cost and elevated production rate, spray drying involves exposure to elevated temperatures and high osmotic pressure, that can lead to a decrease in viability (Rajam & Anandharamakrishnan 2015). Encapsulation by extrusion/external gelation is a simpler method that, even though it enables high cell viability, it produces capsules larger than 500 μm, which can ultimately negatively impact sensorial analyses, impeding successful incorporation into food products (Prisco et al. 2015). Internal gelation, on the other hand, offers the possibility to produce smaller capsules (<100 μm), using a gentler method that is highly efficient in cell viability protection and is more affordable at a laboratory scale, which makes it as one the most promising encapsulation techniques for future applications (Šipailienė & Petraitytė 2018). Both emulsification techniques produce capsules from a water-in-oil (W/O) dispersion (Poncelet et al. 1992; Poncelet et al. 1995), although internal gelation emerged as an alternative to external gelation since it overcomes the difficulty in dispersing calcium chloride (CaCl2; Ca2+ source) in the oil phase, which ultimately produces clumped capsules (Poncelet et al. 1995). In this manner, acidification allows the release of Ca2+ from an insoluble calcium salt (usually calcium carbonate – CaCO3), which interacts with alginate forming an “egg-box”-like structure, hence producing microcapsules (Poncelet et al. 1992; Poncelet et al. 1995).
Ideally, the combination of different strategies of probiotic delivery systems might offer increased effectiveness in providing sufficient quantities of bacteria to guarantee health benefits for the patient. According to our best of knowledge, only two studies approached the issue of delivery systems for A. muciniphila. Van der Ark and colleagues (2017) encapsulated A. muciniphila in a double emulsion having achieved a high viability during in vitro gastric passage, and Marcial-Coba et al (2018) by extrusion
method reporting acceptable storage stability. The lack of strategies for adequate delivery systems or carriers involving A. muciniphila reveals an urgent need for research studies targeting new approaches using proper chemical agents and materials that offer probiotics protection against GI conditions and simultaneously promote their viability and stability during storage.
1.4. Thesis aim
Keystone species A. muciniphila demonstrates a great potential in the prevention and treatment of dysbiosis-associated diseases. Given its sensitivity to oxygen, preservation strategies are paramount for the introduction of this NGP into the health market. The protective effects of some technological solutions for enhancing viability and stability of probiotics, such as Bifidobacterium spp, Lactobacillus spp and some yeasts, upon harsh GI and storage conditions, have been shown. Based on the above rationale, the main goal of the present work was the design and study of technological strategies capable of accomplishing a protective impact on A. muciniphila when exposed to detrimental GI conditions as well as to storage at ambient air.
Considering the main objective of the work, the study was divided into two parts with different specific objectives:
Part I – Phenotypic characterization of A. muciniphila DSM22959 through study of several physiological properties:
i) Study of growth and death kinetics of A. muciniphila DSM22959 exposed to different conditions;
ii) Evaluation of A. muciniphila viability and stability during exposure to simulated GI transit conditions;
iii) Evaluation of A. muciniphila ability to adhere to in vitro intestinal epithelium models.
Part II – Design and study of technological strategies based on freeze-dried formulations and encapsulation.
iv) Design, preparation and freeze-drying of formulations incorporating A. muciniphila therein and evaluation of viability and stability of formulated bacteria under simulated GI transit and throughout aerobic storage under different conditions.
v) Encapsulation of A. muciniphila via emulsification/internal gelation method and evaluation of viability and stability of encapsulated bacteria under simulated GI transit and throughout aerobic storage under different conditions.
Introduction - State of the Art
Growth Akkermansia muciniphila DSM
22959
Part I - Phenotypic Characterization
Experimental Work
Oxygen tolerance Adhesion capacity FCM FFA profile Core-Formulation Core-Formulationsupplemented with starch
Formulation Microencapsulation
Microencapsulation
Part II – Design and study of technological strategies
in vitro simulated GI
conditions Storage conditions
Results & Discussion
Concluding Remarks & Future Work
2.
Material and Methods
2.1. Phenotypic characterization of A. muciniphila DSM22959
2.1.1. Akkermansia muciniphila cultivation under reference conditions
Akkermansia muciniphila strain DSM 22959 from DSMZ collection (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany) was used in the present work. The bacterial strain was kept at -80 ºC in PYGM broth (Table 2.1) with 20 % (v/v) glycerol (Fisher Chemical, UK) until phenotypic analyses and technological studies, based on freeze-dried formulations and encapsulation, were performed.For each analysis and study, A. muciniphila DSM 22959 was reactivated from the frozen state in PYGM broth, in Hungate culture tubes sealed with butyl rubber septa (Bellco Glass, USA), incubated for 17±2 h at 37 °C under anaerobic atmosphere (80 % N2, 10 % H2 and 10 % CO2) in an anaerobic chamber (Whitley DG250 Anaerobic Workstation, UK), with at least two subsequent culturing steps at the same growth conditions.
The PYGM broth was prepared as described in table 2.1 with pH adjusted to 7.2 using 8 N NaOH (Labor Spirit, Portugal). PYGM agar was prepared with addition of 1.5 % (w/v) agar (Liofilchem, Italy). Both PYGM broth and PYGM agar media were then autoclaved at 121 ºC for 20 min. Upon sterilization, the absence of oxygen in the media was verified by its color; if anaerobic conditions prevail a brown color is achieved, if exposed to aerobic atmosphere, the medium becomes pink.
Table 2.1 - Composition of Akkermansia muciniphila reference growth medium (PYGM broth) according to DSMZ.
Ingredients Concentration Manufacturer Trypticase peptone 5 g.L-1 VWR Chemicals, EC
Peptone 5 g.L-1 VWR Chemicals, Belgium
Yeast extract 10 g.L-1 VWR Chemicals, Belgium
Beef extract 5 g.L-1 VWR Chemicals, India
Glucose 5 g.L-1 Sigma-Aldrich, USA
Mucin 0,5 g.L-1 Sigma-Aldrich, USA
K2HPO4 2 g.L-1 Sigma-Aldrich, USA
Tween 80 1 mL.L-1 VWR Chemicals, France
Cystein-HCl 0,5 L-1 Alfa Aesar, Germany
Resazurin 1 mg.L-1 Sigma-Aldrich, USA
Salt Solution 1 40 mL.L-1 NA
Hemin Solution 2 10 mL.L-1 NA
Vitamin K1 Solution 3 200 µL.L-1 NA
dH2O 0,95 L NA
1Salt solution composition according to table 2.2.
2 Hemin solution: 50 mg of hemin (Alfa Aesar, Germany) in 1 mL of 1 N of NaOH and diluted into 100 mL
of dH2O. 3 Vitamin K
1: 0.1 mL of vitamin K1 (Sigma-Aldrich, USA) in 20 mL of 95 % (v/v) ethanol filtered by 0.22 µm
sterile filter (Millipore, USA). NA – Not applicable
Table 2.2 - Composition of salt solution included in Akkermansia muciniphila reference growth medium according to DSMZ.
Ingredients Concentration Manufacturer CaCl2.2H2O 0.25 g.L-1 Carlo Erba Reagents, Italy MgSO4 · 7H2O 0,5 g.L-1 Merck, Germany
K2HPO4 1 g.L-1 Sigma-Aldrich, USA KH2PO4 1 g.L-1 Sigma-Aldrich, USA NaHCO3 10 g.L-1 Merck, Germany
NaCl 2 g.L-1 Labchem, USA
dH2O 1 L NA
2.1.2.
Growth kinetics and cellular characterization
Determination of growth kinetics. To obtain the growth curves, two replicas were performed. For each replica, 45 mL of PYGM broth was inoculated with 5 mL of A.
muciniphila grown according to procedures described in section 2.1.1 and incubated at 37 ºC under anaerobic conditions (80 % N2, 10 % H2 and 10 % CO2) for 24 h. Growth curves were determined by measuring optical density (OD) at 600 nm in a spectrophotometer (UV mini-1240 Shimadzu, Japan), throughout time and simultaneously by quantification of colony-forming units (CFU) of A. muciniphila in some sampling points. For viable cell numbers quantification, ten-fold serial dilutions of bacterial suspension were performed in phosphate buffer saline (PBS x1; VWR, USA). Then, 10 µL of each dilution was spotted, in triplicate, on PYGM agar plates subsequently incubated for 7 d at 37 ºC under anaerobic conditions, and results were expressed as CFU per milliliter.
Cellular morphology and Gram stain. The cellular morphology and Gram test of 17±2 h grown A. muciniphila cells were determined after Gram staining as described by Bartholomew & Mittwer (1952) and examination by optical microscopy at 100x magnification.
2.1.3. Oxygen tolerance
To assess A. muciniphila tolerance to oxygen, fully grown cultures at the early stages of stationary phase (17 ±2 h growth) were exposed to ambient air atmosphere at 37 ºC with and without agitation (140 rpm) over a period of 60 h. A positive growth control was included and consisted in bacterial growth at 37 ºC under anaerobic conditions as described in section 2.1.1. For each sampling point, the total viable cell numbers of A. muciniphila, grown in each atmosphere, were determined by plating ten-fold serial dilutions of bacterial suspension, in triplicate, on PYGM agar plates according to procedures described in 2.1.2. Two replicas were performed for each atmosphere condition tested.
2.1.4. Adhesion assays to intestinal epithelium cells
2.1.4.1. Epithelial Cell Lines
The caucasian colon adenocarcinoma (Caco-2) and mucus secreting human colon adenocarcinoma (HT29-MTX-E12) cells were obtained from the European Collection of Authenticated Cells Cultures (ECACC 8601020 and 12040401, respectively) through Sigma-Aldrich (ECACC, USA) (references 09042001 and 12040401, correspondingly). The cell lines were grown at 37 °C under a humidified atmosphere (95%) in an incubator supplemented with 5 % CO2. Except when stated otherwise, the cells were grown using high glucose (4.5 % (w/v)) Dulbecco’s Modified Eagle’s Medium (DMEM;
